CN116983320A

CN116983320A - Nucleic acid medicine for treating skin fibrosis and preparation method and application thereof

Info

Publication number: CN116983320A
Application number: CN202311158848.2A
Authority: CN
Inventors: 林云锋; 姜玥莹; 李松航; 田陶然
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-09-08
Filing date: 2023-09-08
Publication date: 2023-11-03

Abstract

The invention provides a nucleic acid medicine for treating skin fibrosis, a preparation method and application thereof, and belongs to the field of nucleic acid molecule medicines. The invention firstly provides a tetrahedral framework nanomaterial for treating skin fibrosis, which is prepared by base complementary pairing of nucleotide sequences shown in SEQ ID NO. 5-8. The tetrahedral framework nanomaterial can be used for preparing nucleic acid medicines for treating skin fibrosis. The tetrahedron nanometer material of the invention has the functions of resisting cell death and inflammation, reducing skin fibrosis, and being used in preparing nucleic acid medicine for treating skin fibrosis.

Description

Nucleic acid medicine for treating skin fibrosis and preparation method and application thereof

Technical Field

The invention belongs to the field of nucleic acid molecule medicaments, and in particular relates to a nucleic acid medicament for treating skin fibrosis, a preparation method and application thereof.

Background

Skin is the largest organ of the human body and plays a vital role as the first line of defense against various endogenous and exogenous infectious factors and injuries. Adverse events affect skin homeostasis, making the skin susceptible to a variety of stimuli and toxins. When the intensity or duration of a skin injury exceeds the capacity of tissue repair, skin fibrosis caused by a disregulation of the tissue repair response begins to dominate the repair process. The heterogeneity of skin tissue repair reactions can lead to scar formation, which is the result of pathologically connective skin tissue repair, which can be observed in hypertrophic scars and keloids, while scleroderma or scleroderma occurs in a limited or diffuse type of systemic sclerosis.

Skin fibrosis not only affects aesthetics, but also causes the skin to lose elasticity and activity, and many uncomfortable symptoms appear, seriously affecting work and life. Current methods of treatment of hypertrophic scars and keloids, such as topical corticosteroid injection, surgical revision, cryotherapy, radiation therapy, and even the use of silica gel containing onion extract, cannot fundamentally treat, are prone to recurrence and must be repeated. For scleroderma, there is no generally accepted treatment, steroid hormone, methotrexate injection can be used, but the side effects are large. It can be seen that the existing treatment methods can not achieve ideal curative effects.

In recent years, various novel nucleic acid materials, such as nano-sized material tetrahedral framework nucleic acids (tFNAs), have been developed and widely used in various fields. tFNAs can act as anti-inflammatory and antioxidant agents to promote wound healing in the skin. Previous studies have shown that tFNA can carry mirnas to deliver mirnas into cells or tissues that perform specific functions without a vector. In view of its excellent biosafety and biocompatibility, the tFNA structure is further optimized, and miRNA is further optimized from a cohesive end linkage as part of a tetrahedral structure, improving the delivery efficiency of miRNA. The material is expected to be used for treating fibrotic diseases, in particular pulmonary fibrotic diseases.

Most of the existing methods for carrying miRNA on tetrahedrons need to adhere the sticky end to the vertex or the side arm of the tetrahedron frame, which causes the problems of overlarge material diameter, poor stability, low medicine carrying efficiency and the like.

Further research is needed on how to improve the therapeutic effect of tetrahedra on skin diseases.

Disclosure of Invention

The invention aims to provide a nucleic acid medicine for treating skin fibrosis, and a preparation method and application thereof.

The invention provides a tetrahedral framework nano material for treating skin fibrosis, which is prepared by base complementary pairing of nucleotide sequences shown in SEQ ID NO. 5-8.

Further, the molar ratio of the nucleotide sequence shown in SEQ ID NO. 5-7 to the nucleotide sequence shown in SEQ ID NO.8 is (1-5): 3-15.

Further, the molar ratio of the nucleotide sequence shown in SEQ ID NO. 5-7 to the nucleotide sequence shown in SEQ ID NO.8 is 1:1:1:3.

The invention also provides a preparation method of the tetrahedral framework nano material, which comprises the following steps:

mixing the nucleotide sequences shown in SEQ ID No. 5-8 in a solvent, maintaining the temperature at 90-95 ℃ for 10-20 minutes, and rapidly cooling to 4-10 ℃ and maintaining the temperature for more than 20 minutes to obtain the nucleotide sequence.

Further, the solvent is a TM buffer.

Further, the TM buffer is prepared from 10mM Tris-HCl and 50mM MgCl ₂ Dissolving in water, and adjusting pH to 8.0.

The invention also provides application of the tetrahedral framework nanomaterial in preparing nucleic acid medicines for treating skin fibrosis.

Further, the nucleic acid drug is an anti-cell apoptosis drug.

Further, the nucleic acid drug is an anti-inflammatory drug.

Further, the nucleic acid agent is an agent that inhibits the TGF-beta/Smad pathway.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a novel tetrahedral nano material, which is loaded with miRNA-27a molecules, can resist cell apoptosis and anti-inflammatory activity, can reduce skin fibrosis, can be used for preparing nucleic acid medicines for treating skin fibrosis, and has wide application prospect.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 shows the results of preparation and characterization of BiRDS and tFNA: a is a synthetic schematic diagram of BiRDS; b is an agarose gel electrophoresis result graph of BiRDS and tFNA and a Cy5 and Gelred fluorescence intensity characterization result ImageJ semi-quantitative analysis graph; c is a size distribution and zeta potential result graph of BiRDS and tFNA; d is an AFM morphology analysis result graph of BiRDS and tFNA; e is an agarose gel electrophoresis result diagram of BiRDS incubated for 2 hours at the temperature of 0-10% serum 37 ℃ and an image J semi-quantitative analysis diagram of Cy5 and Gelred fluorescence intensity characterization results; f is an agarose gel electrophoresis result diagram of BiRDS incubated for 0-24 hours at the temperature of 1% serum 37 ℃ and an image J semi-quantitative analysis diagram of Cy5 and Gelred fluorescence intensity characterization result; g is an agarose gel electrophoresis result graph of BiRDS incubated for 0-7 days at a serum-free room temperature of 25 ℃, and a Cy5 and Gelred fluorescence intensity characterization result imageJ semi-quantitative analysis graph; h is a schematic diagram of BiRDS entering cells and being decomposed by enzyme to release miRNA.

FIG. 2 shows the results of in vitro experiments of BiRDS and tFNA cell entry and fibrosis inhibition: a is a cell entering schematic diagram of BiRDS and tFNA; b is semi-quantitative analysis of a cell-entering schematic Cy5 of BiRDS and tFNA at different time points of culture; c is the experimental result of the activity CCK8 of the HaCaT cells of tFNA and BiRDS; d is an immunofluorescence result graph of the expression influence of miRNA, tFNA and BiRDS on alpha SMA in a HaCaT cell fibrosis model established by TGF-beta; e is a western blot experiment (WB) result diagram of the expression influence of miRNA, tFNA and BiRDS on alpha SMA and type I collagen in a HaCaT cell fibrosis model established by TGF-beta; f is an imageJ semi-quantitative analysis chart of alpha SMA expression in D; g is an immunofluorescence result graph of the effect of miRNA, tFNA and BiRDS on the expression of E-cadherein in a HaCaT cell fibrosis model established by TGF-beta; h is an ImageJ semi-quantitative analysis chart expressed by E-cadherin in G; i is an ImageJ semi-quantitative analysis chart of alpha SMA expression in E; J. k is an immunofluorescence result graph and an ImageJ semi-quantitative analysis graph of the effect of miRNA, tFNA and BiRDS on the expression of type I collagen in a HaCaT cell fibrosis model established by TGF-beta.

FIG. 3 is a chart of BiRDS inhibition smad2/3 pathway and focal death pathway: a is the WB result of Fibronectin and smad2/3 analysis of protein expression level; B. c is the IF image and 3D heatmap of fibronectin and smad 2/3; D. e is the semi-quantitative result analysis of WB and IF experiments in A-C; f is a schematic diagram of the influence of BiRDS on different molecules and cell behaviors in the focusing death path; g is BiRDS and tFNA inhibiting protein WB result diagram related to the scorching pathway; H. i is the IF image and 3D heat map of caspase-1 and IL-1β; J-N is a semi-quantitative analysis chart of the expression of the protein related to the apoptosis in the WB and IF experiments in sequence.

FIG. 4 is the experimental results of BiRDS inhibiting skin fibrosis in vivo: a is an in-vivo experimental scheme diagram, comprising the establishment of a fibrosis model, the injection of bleomycin, the detection of molecular biology and the data analysis; b is Masson staining of skin tissue on day 21; c is skin thickness and data analysis chart at days 0, 7, 14 and 21; d is a skin hydroxyproline content analysis chart of the back of the mice in the experiment group on the 21 st day; e is a skin tissue HE staining chart, and the enlarged solid and dotted boxes represent an epithelial structure and a subcutaneous tissue structure respectively; F. g is a fluorescence signal intensity profile detected within 120 minutes of injection of the miR-27a mimetic, biRDS and tFNA.

FIG. 5 shows the results of BiRDS inhibiting bleomycin induced EMT and smad pathways: a is TGF-beta, activates EMT and smad2/3 pathway in animal tissue, and can be reversed by BiRDS; B. c is an image of TGF-beta and alpha SMA expression from an Immunohistochemical (IHC) staining experiment performed on skin tissue; d is a dual fluorescent staining pattern of smad and fibronectin expression levels; e is the index gray value analysis chart in FIG. 5B, F is the index gray value analysis chart in FIG. 5C, and G is the index immunofluorescence intensity gray value analysis chart in FIG. 5D.

Fig. 6 is a diagram of the BiRDS inhibiting the focal death pathway and inflammation in vivo: A. d is the IF result of caspase-1 cleaved in bleomycin group hair follicles; B. e is IHC staining image of IL-1 beta; C. f is an IHC staining image and analysis of bleomycin induced up-regulation of the typical inflammatory cytokine tnfα in skin tissue.

Detailed Description

The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

In the invention, miRNA-27a can be abbreviated as miR27a.

Example 1 preparation of DNA tetrahedron framework nanonucleic acid drug carrying miRNA27a

1. Preparation of BiRDS and tFNA

The nucleotide sequences in the single DNA/RNA strand (ssDNA/ssRNA) are shown in Table 1. All ssDNA/ssRNA was synthesized by Sangon co., ltd., ltd. (Shanghai, china).

Preparation of tFNA: 4 ssDNA (S1-4-T, 1. Mu.M) at equimolar concentration were added to TM buffer (10 mM Tris-HCl and 50mM MgCl) ₂ Dissolved in water and pH adjusted to 8.0 with hydrochloric acid), then the whole solution was heated to 95 ℃ for 10 minutes and immediately cooled in a thermal cycler at 4 ℃ for 20 minutes to complete the denaturation and assembly process to give tFNA.

Preparation of BiRDS: three single stranded DNA/RNA (S1-3-BiRDS, 1. Mu.M) and miRNA-27a (3. Mu.M) were added to TM buffer (10 mM Tris-HCl and 50mM MgCl) at equimolar concentrations ₂ Dissolve in water and adjust pH to 8.0 with hydrochloric acid), then heat the entire solution to 95 ℃ for 10 minutes and immediately cool in a thermal cycler at 4 ℃ for 20 minutes to complete the denaturation and assembly process, resulting in a BiRDS tetrahedral structure. FIG. 1A is a schematic diagram of the synthesis of BiRDS.

TABLE 1 nucleotide sequences of the single DNA/RNA strand

2. Structural characterization of BiRDS and tFNA

To confirm successful synthesis of BiRDS and tFNA, nucleic acids were isolated using GelRed-stained 2% Agarose Gel Electrophoresis (AGE) and running at 120V for 30 minutes in an electrophoresis apparatus (Bio-Rad, usa). To verify the nanostructure of BiRDS and tFNA, their morphology was observed using an Atomic Force Microscope (AFM) (kyoto island in japan). The dimensions and zeta potential of the BiRDS and tFNA were measured using the Zetasizer Nano ZS system (england malva). And (3) grafting Cy5 (Cy 5-S1-T) on the S1-T chain of the tFNA during agarose gel electrophoresis detection, then preparing the tFNA (Cy 5-tFNA) according to the method, or grafting Cy5 (Cy 5-miRNA-27 a) on miRNA-27a, and then preparing the BiRDS (Cy 5-BiRDS) according to the method.

FIG. 1B is a graph of the agarose gel electrophoresis results of BiRDS and tFNA and a graph of the semi-quantitative analysis of Cy5 and Gelred fluorescence intensity characterization results of ImageJ, and the graph of the tFNA agarose gel electrophoresis results, the graph of the BiRDS agarose gel electrophoresis results, and the Cy5 and Gelred fluorescence intensity characterization results are sequentially shown from left to right. Agarose gel electrophoresis (FIG. 1B) confirmed the successful synthesis of tFNA and BiRDS, revealing the molecular weights of the intermediate, tFNA and BiRDS, about 30 to 180bp. Notably, single-stranded DNACy5 showed a weaker fluorescence intensity than the single-stranded DNA supported on the frame at the same electrophoresis time, indicating better stability of the single-stranded DNA (in red dashed box) with the single-stranded DNA exposed on the frame. In the chart of the result of BiRDS agarose gel electrophoresis of FIG. 1B, r is the structure formed by the synthesis of miRNA-27a, S123+r, S123+2r and S123+3r respectively represent S1-BiRDS, S2-BiRDS and S3-BiRDS chains with equimolar concentration and a miRNA-27a mixed system with 1-time, 2-time or 3-time molar concentration. The single-stranded miR27a was attached to the tetrahedral framework nucleic acid in proportion and showed the same trend of fluorescent growth.

AFM morphology analysis of tFNA and BiRDS (fig. 1D) revealed triangular nanoscale particles with a diameter of 10nm, consistent with the size distribution determined by dynamic light scattering (fig. 1C). Both tFNA and BiRDS were negatively charged and the average zeta potential was-7.70 mV and-8.72 mV, respectively. This property favors the rejection of ROS and the reaction with certain functional proteins, which is also the basic principle of electrophoretic separation.

The beneficial effects of the present invention are demonstrated by specific test examples below.

Serum stability of test example 1, biRDS and tFNA

1. Experimental method

To verify stability and storage capacity of tFNA and BiRDS in serum, the Cy 5-linked BiRDS prepared according to example 1 was incubated with serum of different concentrations and tested for serum stability under 3 reaction conditions: (1) Incubating in 0-10% strength fetal bovine serum (FBS, corning company, NY) at 37 ℃ for 2 hours, (2) incubating for 0-24 hours at 37 ℃ in 1% fetal bovine serum; (3) Incubation is carried out for 0-7d under the condition of 25 ℃ in 0% fetal bovine serum. The solvent for adjusting the concentration of the fetal bovine serum is PBS buffer.

After incubation, the samples were used in the presence of 5mM Mg ²⁺ The final product was detected by performing a PAGE experiment in 0.5 xTAE buffer. In order to ensure stability of the final product during the detection, the present invention cools the liquid medium in advance and places it in an ice bath. The gel was visualized using a gel and blot imaging system (bamboole Syngene, india).

2. Experimental results

The present invention incubates BiRDS with 0-10% FBS for 2 hours at 37℃to simulate the in vivo environment and ideal growth conditions of cells. The fluorescence signal of Cy5 detected by PAGE indicated that as FBS concentration was further increased to 10%, the bisds retained the non-serogroup content of 70%, indicating that bisds had good stability in vivo for 2 hours. FIG. 1F illustrates that BiRDS has good stability over 24 hours of incubation in 1% FBS, and that BiRDS was incubated in 1% FBS to simulate the culture environment, and after 24 hours, the fluorescence intensity of Cy5 was maintained at 85% at 0 hours. This ensures that the BiRDS remains in its original form for 24 hours, thereby providing a reliable measure of the number of particles entering the cell, ensuring that the nucleic acid drug is able to enter the cell effectively over a period of time. The storage conditions of the biologics are extremely stringent to maintain their efficacy, which conditions result in high health costs for cryopreservation during cold chain logistics, and FIG. 1G shows that BiRDS exhibits extremely high stability at room temperature (25 ℃) for at least 7 days, and maintains at least 75% of the viability of miR-27a, which is easy to store, facilitating its use in complex environments and in Schottky regions.

Test example 2, cell uptake and cell treatment of BiRDS and tFNA

1. Experimental method

HaCaT cells (BNCC, henan, china) were cultured in RPMI-1640 medium containing 10% FBS and 1% (v/w) penicillin/streptomycin (Hyclone, USA) at 37℃and 5% CO ₂ Is cultured in a humid incubation environment. FBS in the flask was gradually reduced to 1% for 3 hours prior to incubation of the cells with the nucleic acid drug to reduce protein corona formation and prepare for the next procedure.

For cell uptake experiments, haCaT cells seeded in confocal dishes were incubated in medium containing different concentrations of nucleic acid drug (Cy 5-tFNA 250nM, cy5-BiRDS250nM, cy 5-S1-T750 nM, cy5-miRNA-27a 750 nM). After 6-12h incubation, samples were fixed with 4% paraformaldehyde for 30min, and nuclei and cytoplasm were labeled with DAPI and Fluorescein Isothiocyanate (FITC), respectively. Sections were washed three times with PBS for 5 minutes between adjacent steps. Finally, an image was captured using a confocal laser microscope (FV 3000, olympus japan). Cy5-tFNA, cy5-BiRDS, cy5-S1-T and Cy5-miRNA-27a were prepared as described in example 1.

2. Experimental results

The interaction between BiRDS and HaCaT cells is critical for inhibiting fibrosis. BiRDS enters the complex intracellular environment through a small-cell protein mediated endocytic pathway and is targeted by RNase H recognizing the DNA and RNA hybridization region (containing purple hydrogen bonds). The three-dimensional structure of the BiRDS is then converted into a two-dimensional planar structure. In addition, miRNA-27a molecules are released into the cytoplasm to perform different functions. To compare the cellular uptake efficiency of BiRDS with tFNA and related single strands, the present invention uses immunofluorescence to detect the uptake of miRNA-27a by inserting 250nM fluorescent Cy5-tFNA, cy5-BiRDS and fluorescent single strands with the same concentration of Cy 5. As shown in fig. 2A and 2B, biRDS localizes in the cytoplasm in the same manner and exhibits almost the same uptake efficiency as tFNAs. After 12 hours incubation with HaCaT cells, the fluorescence intensity of Cy5 in both the BiRDS and tFNA groups remained at the same level and showed no statistical difference, although the time of BiRDS entry into the cells appeared to be slightly later than tFNA. Both exhibit higher cell permeability. In FIG. 2B, S is Cy5-S1-T, T is Cy5-tFNA, m is Cy5-miRNA-27a, and Bi is Cy5-BiRDS.

Test example 3 cell viability measurements of BiRDS and tFNA

1. Experimental method

To examine the effect of nucleic acid drugs on HaCaT cells, a cell count kit-8 (CCK 8) assay was performed. Different concentrations (31.3, 62.5, 125, 250 and 500 nM) of tFNas and BiRDS were incubated with cells in 96-well plates for 24 hours, then 10. Mu.L of CCK-8 solution was added to the wells and measured at an absorbance of 450 nM. tFNAs and BiRDS were prepared as described in example 1.

2. Experimental results

The invention uses CCK8 experiments to detect cell proliferation and viability. HaCaT cells showed increased proliferation capacity after incubation with tFNA and BiRDS at gradient concentrations of 31.3nM to 500nM (fig. 2C), indicating the non-toxicity of both nucleic acid drugs. In addition, tFNA and BiRDS showed the strongest ability to enhance cell viability at concentrations of 250nM and 125nM, respectively. Thus, the present invention selects these concentrations as the optimal concentrations for subsequent experiments.

Test example 4 Immunofluorescence (IF) assessment of BiRDS and tFNA

1. Experimental method

After HaCaT cells were seeded on confocal dishes and the density was close to 60%, related modeling and treatments were performed:

in addition to the control group (normal HaCaT cells, ctrl), the other four groups were treated with 5ng/mL TGF- β (transforming growth factor β) (MedChemExpress, monmouth Junction, NJ) for 24 hours to model skin fibrosis, and 3 groups were incubated with nucleic acid drug (miRNA-27a 375nM,tFNA250 nM,BiRDS125nM) added simultaneously with TGF- β, respectively.

The harvested samples were fixed with 4% paraformaldehyde for 30min, then with 0.5% Triton X-100 for 10 min, and with 5% sheep serum for 1 hr. Various concentrations of diluted primary antibodies were incubated overnight at 4 ℃ (α -smooth muscle actin (α -SMA), 1:500; fibronectin, 1:300; collagen I,1:300; e-cadherin, 1:500; smad2/3,1:300; cleaved caspase-1, 1:250; il-1β, 1:150), and then the corresponding fluorescent secondary antibodies FITC and DAPI (martial Servicebio, china) were incubated with the samples for 1 hour, 30 minutes and 10 minutes, respectively. Similarly, confocal laser microscopy was used to capture fluorescent signals, while fluorescence intensity measurements of one protein selected the same parameters.

2. Experimental results

Tissues contribute to resilience and strength by maintaining extracellular matrix (ECM) secreted by mesenchymal cells and other matrix components. The over-expressed TGF- β will promote the development of epithelial cells to fibroblasts, known as epithelial-mesenchymal transition (EMT). The invention herein uses transforming growth factor-beta as an actuator to model fibrosis in vitro. After 24 hours of addition of 5 ng/mLTGF-beta to the medium, the mesenchymal marker alpha-SMA was upregulated 3.2-fold. However, it was reversed when exposed to 250nM tFNA and 125nM BiRDS. Compared to the tFNA group, the expression level of α -SMA was lower, and the BiRDS restored the extent of EMT to almost the same level as the control group (fig. 2D-2F).

E (epithelial) -cadherin, known as a "cell adhesion" glycoprotein, is a single transmembrane protein responsible for intercellular cross-talk and maintains the polarity and integrity of epithelial cells. E-cadherin silencing has long been recognized as a key driver affecting epithelial cell separation from one another and leading to a dramatic loss of cell polarity. The factor may be regulated in content or location by various fibrotic cytokines, including TGF- β. In the IF test, the present invention observed that after 5 ng/mLTGF-beta treatment, the fluorescent signal of E-cadherin became weaker and blurry, indicating that its downregulation and dislocation was due to EMT. These changes were reversed after tFNA and BiRDS treatment. Proteins returned to the membrane and showed a clearer, slightly lighter fluorescent signal than the control group, which helped to maintain the polarity and stability of HaCaT cells (fig. 2G and 2H).

BiRDS reduces expression of matrix proteins and inhibits the smad2/3 pathway. In chronic fibrotic disease, TGF- β favors mesenchymal transformation of epithelial cells and promotes transformation into myofibroblast-like cells. In the late stages, excess extracellular matrix (ECM), including type I collagen, fibronectin, elastin, and proteoglycans, increases due to increased synthesis and decreased degradation in fibroblasts. In the skin fibrosis model of the present invention, 5ng/mL TGF-beta promoted expression of type I collagen and fibronectin in HaCaT cells at 1.6 and 4.3 fold, respectively (FIGS. 3B and 3D). Although 250nM tFNA mainly reversed pathogenic synthesis of both proteins and the same concentration of miR-27a molecules exerted their effect by entering the cells, as previously reported, biRDS as a more efficient version of tFNA and a protective agent for delivery of miR-27a achieved excellent inhibition at low concentrations of 125nM (FIGS. 2J and 2K).

TGF-beta is closely related to fibrosis and is the basis for regulating ECM gene expression. Its downstream molecules, including Smad2/3, act as the basis and key mediators in fibrosis occurrence, becoming an effective target for anti-fibrotic therapy for preventing tissue fibrosis. Based on WB and IF results, smad2/3 was down-regulated when HaCaT cells were treated with tFNAs and BiRDS simultaneously (fig. 3A, 3C and 3E). Although tFNAs showed a powerful therapeutic effect, biRDS further enhanced the inhibition response and optimized the results at nearly the same level as the control group. These results indicate that BiRDS is a stronger nanoscale therapeutic that inhibits the TGF- β/Smad pathway and treats skin fibrotic diseases.

Furthermore, tFNA showed potent inhibition of the focal pathway (fig. 3H and 3I), but BiRDS inhibited the downstream pathway protein more strongly due to interaction with miRNA-27 a.

Test example 5 Western Blot (WB) assay of BiRDS and tFNA

1. Experimental method

After treatment according to the method described in test example 4, cells were harvested using a whole protein extraction kit (KeyGen biotechco., ltd., south kyo, china) to obtain total protein measurements. Target proteins were separated using 10-12% Sodium Dodecyl Sulfate (SDS) -PAGE. Proteins of a specific molecular weight were transferred to PVDF membranes and immediately blocked with 5% Bovine Serum Albumin (BSA) for 1 hour. After incubation overnight with primary antibodies [ anti- α -SMA (1:1000), anti-E-cadherin (1:10000), anti-collagen I (1:1000), anti-fibronectin (1:1000), anti-TNF- α (1:800), anti-NLRP 3 (1:1000), anti-lytic caspase-1 (1:1000), anti-GSDMD (1:1000), anti-IL-18 (1:1000), anti-IL-1β (1:1000) ] at 4 ℃, PVDF membranes were incubated with the appropriate secondary antibodies (1:3000) for 1 hour. Exposure signals were acquired from the gel and blot imaging system for subsequent semi-quantitative analysis in ImageJ.

2. Experimental results

The results show that: both alpha-SMA and type I collagen increased after 5ng/mL TGF-beta treatment, and BiRDS and tFNA inhibited the decrease in both levels (FIG. 2I). TFNA and BiRDS inhibited fibronectin and Smad2/3 to different degrees, with the same trend as the results in test example 4. The classical focal death pathway recruits NLRP3 and forms inflammatory corpuscles with caspase-1. Activated caspase-1 promotes cleavage and pore formation of GSDMD, thereby promoting exudation of mature IL-18 and IL-1β (FIG. 3F). After miR27a treatment, NLRP3 expression was down-regulated compared to TGF- β group (fig. 3G and 3J). The unique structure of BiRDS can be used as a delivery carrier for transferring miRNA-27a into cells, and the stability of a single chain can be maintained even if the miRNA with the same concentration is added. In the BiRDS group, almost all of the pyro-proteins were inhibited to the level of the control group. In contrast, GSDMD, caspase-1, IL-18 and IL-1β reached lower levels in WB (FIGS. 3J-3N), indicating that tFNA and BiRDS are anti-scorch drugs with high application potential.

Test example 6, animal model building and grouping

1. Experimental method

All animal experiments were performed according to the animal ethics committee of the university of Sichuan Hua Xi oral medical college. 60 BALB/c mice (6-8 weeks old, 18-22 g) were purchased from Gemphamatech Inc. (Nanj, china) and kept in a 12/12h light/dark cycle. They are used as animal models of fibrosis, and the specific model construction method is as follows: after 7 days of adaptive feeding, mice were injected with 0.1mL of 0.5mg/mL bleomycin hydrochloride (B) on specific areas of the dorsal skin for 3 weeks (once every two days over a period of 3 weeks). Over the next 3 weeks, the mice were divided into six groups: control group, B, B+mi R27a, B+T, B+125nM BiRDS and B+250nM BiRDS, and nucleic acid drug (last day of bleomycin injection, beginning with nucleic acid drug). The drug volumes were 100 μl, miR27a concentration was 750nM, tFNA (T) concentration was 250nM, biRDS concentrations were 125nM and 250nM. Skin tissue (harvested on days 0, 7, 14 and 21, respectively) and viscera (harvested on day 21) were harvested for tissue analysis by injection of 3 weeks of nucleic acid drug (injected every two days over a period of 3 weeks). miR27a is miRNA-27a, T is tFNA, and miRNA-27a, tFNA and BiRDS are as described in example 1.

Skin thickness and histopathological analysis: dehydrated and paraffin-embedded tissues were sectioned into 4 μm sections. The skin was flattened and sectioned longitudinally to observe a lesion in the center of the tissue using an FSX100 microscope (olympus, tokyo, japan). Skin fibrosis was assessed by measuring the vertical epithelial thickness of the blue fibrotic fraction after Masson staining. HE staining was used to observe histological changes of skin and viscera. The dried sections were immediately placed in xylene, dewaxed for 5-10 minutes, and placed in 100% -70% alcohol until the alcohol was washed with water. After staining with hematoxylin and eosin, the tissue was dehydrated. For ease of observation and storage, neutral balsam was dropped on paper towels and baked.

Immunohistochemical analysis: for immunohistochemistry, the remaining sample sections were incubated overnight in diluted primary antibody at 4 ℃, including mouse anti-TGF- β. Specific dilutions of anti-rabbit secondary antibodies (1:600, USA) were used for 20min at 37 ℃. After development, washing, dehydration and mounting, the sections were imaged using an optical microscope.

Distribution of BiRDS and tFNA in vivo: in order to study the distribution, stability and absorption rate of the injected drug, the present invention performed in vivo imaging experiments after in vivo injection of Cy5-BiRDS (250 nM), cy5-tFNA (250 nM, cy5 in S1-3) and Cy5-mi27a (750 nM) in mice. After various time periods (0, 15, 30, 60 and 120 minutes), mice were anesthetized with isoflurane and imaged using an in vivo imaging system (IVIS Spectrum). After 120 minutes, mice were sacrificed to separate organs such as skin, heart, liver, spleen, lung, and kidney that were simultaneously exposed.

2. Experimental results

2.1BiRDS reduced fibers in the epithelium and maintained the epithelial structure

In the last three weeks of treatment, the invention collected skin tissue in the back every 7 days and viscera on day 21 for detection. Skin thickness is often the primary endpoint of bleomycin-induced fibrosis, and can be specifically labeled with Masson trichromatography (fig. 4B). The present invention measured the height from epidermis to subcutaneous tissue in three random non-overlapping pictures of each group, with the average measurement of bleomycin (B) group dermis thickness increasing from 334.2 μm to 394.4 μm over time (fig. 4C). On day 21, the skin thickness was reduced in all treatment groups. BiRDS (125 nM) and tFNA (250 nM) showed the same effect as 750nM miR-27a. The thinnest skin tissue was observed in the 250nM BiRDS group. Experimental results of hydroxyproline evaluation provide further verification of the fibrosis content. Hydroxyproline is formed from the posttranslationally hydroxylated amino acid proline, which is primarily limited to collagen. Fig. 4D shows the hydroxyproline content at the end of the experiment and shows a similar trend to skin thickness. Group B reached 0.64 μg/mg, three times more than the control group, while the 250nM BiRDS reversed its effect to 0.20 μg/mg.

During injection, the experimenter clearly felt that the skin of the mice became more rough and stiff, and HE staining was performed in order to observe the microstructure of the skin tissue (fig. 4E). The solid line box and the dashed line box represent the features and changes of the epithelial and subcutaneous tissues, respectively. In the control group, the present invention observed a large number of dermal papillae, normal glandular structures, abundant subcutaneous adipose tissue and capillaries. However, the severe fibrotic process causes the glandular, fatty and dermal papillae to disappear, with the dilated blood vessels increasingly occupying additional sites in the subcutaneous tissue. Injection of 750nM miR-27a and 125nM BiRDS restored only a portion of the structure, whereas 250nM tRNA and BiRDS showed significant therapeutic effects.

2.2BiRDS maintained the stability of miR-27a and demonstrated skin targeting

To examine metabolism in various organs after subcutaneous injection of drug, the present invention tested the distribution of certain points with IVIS spectroscopy within 120 minutes (fig. 4F). The isosteric drug was injected into bleomycin-induced fibrosis mice. A similar initial strength was ensured immediately after injection. After 15 minutes, the fluorescence intensity of miR-27a drops sharply and remains at a lower level for the last 90 minutes. In contrast, higher concentrations of BiRDS remained in skin tissue and slowly declined to lower levels at 120 minutes, but still higher than bare miR-27a. The degradation of tFNA is relatively uniform compared to the other two drugs, probably because of their lower absorption rate. BiRDS protects the stability of the DNA/RNA single strand and can target organs. The invention showed the lowest fluorescence in the miR-27a group and the strongest fluorescence in the BiRDS-injected skin, indicating its greater stability and higher skin targeting ability (fig. 4G). In addition, weak Cy5 fluorescence was observed in the liver at 120 minutes, indicating that the drug was metabolized in the liver, as previously reported by our group. In our experiments, biRDS showed no significant toxicity in organs and very strong biocompatibility.

2.3BiRDS inhibits the TGF-beta/smad pathway in vivo

During bleomycin-stimulated pathology, latent TGF- β molecules are initiated by a variety of proteases, such as plasmin and metalloproteinases. TGF-. Beta.is also a potent inducer of epithelial cell to myofibroblast cell, characterized by expression of alpha-SMA (FIG. 5A). More and more myofibroblasts express an excess of ECM, while the excess protein is under-decomposed. IHC results indicate that the TGF- β content in the bleomycin group is increased approximately 2.3-fold (FIGS. 5B and 5E). Surprisingly, all treatment groups showed significant therapeutic effects. Naked miR-27a reduced the content to 1.4 times that of the control group, while 250nM BiRDS kept it at a minimum level, almost identical to the control group. tFNA and 250nM BiRDS have excellent curative effect in inhibiting alpha-SMA; the latter group was the most potent, reaching 1.2 times that of the non-fibrotic group (fig. 5C and 5F). BiRDS at 250nM inhibited both fibronectin and Smad proteins (FIGS. 5D and 5G)

2.4BiRDS inhibit Pyropysis-related proteins and TNF-alpha

The in vitro experiment of the invention shows that BiRDS carrying miR-27a inhibits the focal sagging pathway. In order to evaluate the regulation of BiRDS in vivo pathology, immunofluorescence and IHC experiments were performed on cleaved caspase-1 and IL-1β, and semi-quantitative analysis was performed on target proteins. Bleomycin-induced inflammation and fibrosis promoted strong expression of cleaved caspase-1 in hair follicles, sebaceous glands and subcutaneous tissue (fig. 6A and 6D). The expression of the downstream inflammatory cytokine IL-1β varies in the same manner. In contrast to tFNA, each BiRDS particle contains three NLRP 3-targeting miR-27a molecules that mediate the typical focal sagging pathway. Thus, biRDS may inhibit each step of the pyrosis cascade pathway with the highest efficiency in the four groups. IL-1β, the last loop of the pyrosis pathway, is a cytokine secreted by GSDMD pores, which promotes inflammatory responses. The expression level of bleomycin group IL-1β was increased three times and the BiRDS reduced its level slightly above the first group (fig. 6B and 6E). The present invention also detects the expression of tumor necrosis factor alpha (TNF-alpha), which is a key driver of fibrosis. This factor strongly affects the amplification and persistence of tissue damage and fibrosis. BiRDS also inhibited TNF- α expression most effectively at 250nM (FIGS. 6C and 6F), indicating that BiRDS has greater efficacy than tFNA in inhibiting pyrosis and other inflammatory factors, improved anti-pyropic and anti-inflammatory activity, and reduced fibrosis.

In summary, the invention provides a novel tetrahedral nanomaterial, which carries miRNA-27a molecules, and the tetrahedral nanomaterial can resist apoptosis and anti-inflammatory activity, can reduce skin fibrosis, can be used for preparing nucleic acid medicines for treating skin fibrosis, and has wide application prospect.

Claims

1. A tetrahedral framework nanomaterial for treating skin fibrosis, characterized in that: it is obtained by base complementary pairing of nucleotide sequences shown in SEQ ID NO. 5-8.

2. The tetrahedral framework nanomaterial of claim 1, wherein: the mol ratio of the nucleotide sequence shown in SEQ ID NO. 5-7 to the nucleotide sequence shown in SEQ ID NO.8 is (1-5): 1-5: 3-15.

3. The tetrahedral framework nanomaterial of claim 2, wherein: the molar ratio of the nucleotide sequence shown in SEQ ID NO. 5-7 to the nucleotide sequence shown in SEQ ID NO.8 is 1:1:1:3.

4. A method for preparing a tetrahedral framework nanomaterial according to any of claims 1 to 3, characterized in that: it comprises the following steps:

5. The method of manufacturing according to claim 4, wherein: the solvent is a TM buffer.

6. The method of manufacturing according to claim 5, wherein: the TM buffer is prepared from 10mM Tris-HCl and 50mM MgCl ₂ Dissolving in water, and adjusting pH to 8.0.

7. Use of a tetrahedral framework nanomaterial according to any of claims 1 to 3 for the preparation of a nucleic acid drug for the treatment of skin fibrosis.

8. Use according to claim 7, characterized in that: the nucleic acid medicine is an anti-cell apoptosis medicine.

9. Use according to claim 7, characterized in that: the nucleic acid drug is an anti-inflammatory drug.

10. Use according to claim 7, characterized in that: the nucleic acid drug is a drug for inhibiting TGF-beta/Smad channels.